CA1232850A - Purification of superoxide dismutase - Google Patents
Purification of superoxide dismutaseInfo
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- CA1232850A CA1232850A CA000472274A CA472274A CA1232850A CA 1232850 A CA1232850 A CA 1232850A CA 000472274 A CA000472274 A CA 000472274A CA 472274 A CA472274 A CA 472274A CA 1232850 A CA1232850 A CA 1232850A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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Abstract
ABSTRACT
A process for purifying the enzyme, superoxide dismutase, by passing an aqueous solution containing the enzyme, which is free from hemoglobin, through a metal chelating affinity chrom-atographic column, for example an immobilized copper ion column, and eluting an enzyme solution having increased specific activ-ity.
A process for purifying the enzyme, superoxide dismutase, by passing an aqueous solution containing the enzyme, which is free from hemoglobin, through a metal chelating affinity chrom-atographic column, for example an immobilized copper ion column, and eluting an enzyme solution having increased specific activ-ity.
Description
:lZ32850 PURIFICATION OF SUPER OXIDE DECIMATES
This invention relates to a process for the purifica-lion and production of the enzyme commonly referred to as super oxide decimates (SOD). More particularly, the invent lion relates to a process for the purification and production of super oxide decimates by use of a metal chelating cremate-graphic procedure. The invention is particularly suitable for use in the purification of super oxide decimates from hut man outdated red blood cells.
The invention as claimed herein is a process for puff-lying super oxide decimates which comprises subjecting an aqueous solution containing super oxide decimates which is free from hemoglobin to a metal chelating affinity chromatographic procedure and recovering an aqueous solution containing super-oxide decimates having increased specific activity of super-oxide decimates.
The aqueous solution containing super oxide decimates, which is free from hemoglobin, to be used as starting material, may be conveniently obtained, if desired, from outdated human red blood cells. Such cells, preserved in a suitable medium, such as a citrate-phosphate-dextrose-adenine medium, may be lucid by means of deionized water. The Lucite so obtained may be freed from hemoglobin in a variety of ways, such as a) past sage through an anion exchange chromatographic column using, for example, a cross-linked agrees derivative such as deathly-*
amino ethyl (DEAR) - Suffers, or b) a precipitation technique or c) an affinity technique.
It is preferred to use an anion exchange chromatographic column for the preliminary purification procedure to remove hemoglobin. Any suitable anion exchanger may be used, such as a cross-linked agrees derivative, for example DEAR - Suffers, '~''``"~
* Trade Mark 328S~
in conjunction with an appropriate equilibration buffer which may be for example potassium phosphate at pi 6.4. The condo-lions for separating the super oxide decimates from the hero-glob in on the anion exchange column by elusion may be varied considerably. A buffer with a molar concentration of 0.1M or higher and with a pi of about 5 or higher is a suitable buff for for elusion of the super oxide decimates. Such buffers may be, for example, 0.1M potassium phosphate solution used alone or 10mM potassium phosphate containing either elm sod-I'm chloride or elm potassium chloride. Operation of the column at a temperature of about 4C and at a flow rate of about 150 ml/hour provides an equate in the form of an aqueous solution containing super oxide decimates which is suitable for use in the subsequent metal chelating affinity chromatographic procedure.
As an alternative method for providing the starting mat-trial containing super oxide decimates, it is possible to sub-jet red blood cells to a saline wash followed by a tangential flow ultrafiltration system and rupture of the cells hypotoni-gaily before the Lucite is applied to the anion exchange got-urn. Thus, the red blood cells may be washed with about 10 volumes of saline using a tangential flow ultrafiltration soys-them. The saline wash is carried out twice followed by concern-traction of red blood cells using the same ultrafiltration soys-them. Cells are then ruptured, hypotonically, by the addition of 3 volumes of distilled water. The cell membranes are no-moved from the soluble proteins using the ultrafiltration apt pyrites and filtered Lucite is then applied to the anion ox-change column. The ultrafiltration process removes the bulk of the plasma proteins present in red blood cell concentrate and thus increases the efficiency of the process to provide 1232~350 the starting material in the form of an aqueous solution con-twining super oxide decimates. Another variation of the ultra-filtration process that can be used is the removal of insoluble material from red blood cell lusts by filtration without prior washing of the cells with saline.
The metal chelating affinity chromatographic procedure may be carried out on a suitable column wherein the chelated metal is attached to an insoluble matrix. A suitable matrix may be a cross-linked agrees derivative, a derivative from vinyl polymers or a derivative from cellulose or silica or polyacrylamides. Suitable cross-linked agrees derivatives may be, for example, iminodiacetate-Sepharose 6B or ethylene-diamine-Sepharose or tris-(carboxymethyl)ethylenediamine-Seph-arose. Of these, it is preferred to use iminodiacetate-Sepha-rose 6B as the column of choice. A suitable matrix as a don-ivative from vinyl polymers may be iminodiacetate-Fractogel.
Other matrix columns may be prepared from an iminodiacetate-linked derivative of cellulose, polyacrylamide, silica or sync Thetis polymeric supports.
It is to be understood that the commercially available *
product known as iminodiacetate-Sepharose 6B is a beaded age-rose gel consisting of the chelating agent, iminodiacetic acid, coupled to agrees after epoxy activation, the latter introduce in some cross-linking into the matrix. The product thus has bis-(carboxymethyl)-amino groups coupled to agrees via long, stable, hydrophilic spacer arms as shown by the formula:
agarose-O-CH2CHOHCH2O(CH2)4OCH2CHOHCH2N(CH2COOH)2 The metal to be immobilized on the column is preferably copper and the immobilization procedure may be carried out by applying an aqueous solution of a copper salt to the column.
* Trade Mark A variety of copper salts may be used, such as copper sulfite, copper chloride and copper nitrate. A preferred copper salt is copper sulfite and a 50mM aqueous solution of copper sulfite may be applied to a column of iminodiacetate-Sepharose 6B which has previously been equilibrated with a buffer of loom poles-slum phosphate (pi 6.4) containing Old sodium chloride. A
buffer having a pi from about 6 to about 8 provides suitable conditions for binding the super oxide decimates to the copper chelating column. When the metal chelating affinity column has been equilibrated, the aqueous solution containing super-oxide decimates, prepared as starting material, may be applied to the immobilized copper column. Subsequent elusion provides an aqueous solution containing highly purified super oxide disk mutate showing a specific activity of about 3500 unit/mg. As a suitable eluant, there may be used 20mM sodium citrate buff for at pi 5.0 or 20mM sodium citrate buffer at pi 5.0 contain-in lo sodium chloride.
It will be appreciated by those skilled in the art that other suitable columns and other copper salts, with appropriate buffers, may be used to provide the immobilized copper cheat-in affinity column on which the aqueous solution of super oxide decimates might be applied and thereafter eluded to provide highly purified super oxide decimates.
The process of this invention is particularly valuable when the copper chelating column is prepared from an aqueous solution of copper sulfite applied to a column of iminodiace-tate-Sepharose 6B. The process is further enhanced when the starting material to be used on such a copper chelating aft finite column is an aqueous solution containing super oxide disk mutate which has been obtained as an equate from a cremate-graphic column containing diethylaminoethyl (DEAR) - Suffers ~Z3Z850 to remove hemoglobin from human red blood cell Lucite.
Thus, as a further feature of the invention, as claimed herein there is provided a process for purifying super oxide decimates which comprises subjecting human red blood cell lye sate to an anion exchange chromatographic procedure, wherein the anion exchanger is DEAR - Suffers, to provide an aqueous solution containing super oxide decimates free from hemoglobin and then subjecting the aqueous solution to an immobilized copper ion chromatographic procedure, wherein the immobilized copper ion is prepared from an aqueous solution of a copper salt, such as copper sulfite, copper chloride or copper nix irate on a column of iminodiacetate-Sepharose 6B, and then eluding the column to provide an aqueous solution containing super oxide decimates having increased specific activity of super oxide decimates.
The invention is illustrated by, but not limited by, the following Examples.
EXAMPLE I
The following example describes a procedure for purify-in super oxide decimates (SOD) by chromatography on a column of immobilized copper ions.
Outdated red blood cells in suspension (15 ml), pro-served in citrate-phosphate-dextrose-adenine, were lucid with an equal volume of deionized water. The Lucite so obtained was then applied to a 5 cm x 4 cm column of DEAR - Suffers equilibrated with 10mM potassium phosphate buffer (pi 6.4) at 4C and at a flow rate of 150 ml/hour. The same temperature and flow rate were maintained throughout the remainder of the chromatographic procedure. After the Lucite had been applied, the column was washed with approximately 250 ml of equilibria-lion buffer. Partially purified SOD was then eluded from the ~232~350 DEAR - Suffers column with the same equilibration buffer containing elm sodium chloride thereby providing an aqueous solution of partially purified SOD.
A column (5 cm x 2 cm) of iminodiacetate-Sepharose 6B
was activated with 50mM aqueous copper sulfite solution to approximately 3/4 of the bed volume to give an immobilized copper ion column. Prior to application of the partially pun-flied SOD solution, tide immobilized copper ion column was equip liberated with 10mM potassium phosphate buffer pi 6.4) contain-in 0.1M Nail. The aqueous solution of partially purified SOD,eluted from the DEAR - Suffers column, was applied to the imp mobilized copper ion column and thereafter washed through with 10mM potassium phosphate buffer containing lo Nail. After the effluent reached an absorbency of zero, at 280 no, the column was washed with 50 ml of 10mM potassium phosphate buffer (pi 6.4). Purified SOD was eluded from the copper ion chelates column with 20mM sodium citrate buffer (pi 5.0). Enzyme anti-viny (Holland et at., Anal. Become. 1983, 135, 282) and pro-loin determinations (Lowry et at., J. Blot. Chum. 1951, 193, 265) indicated that the specific activity of the SOD increased about 270-fold as a result of this chromatographic procedure on copper ion iminodiacetate-Sepharose 6B. Recovery of enzyme activity, following this procedure, ranged from 75% to 80% of the total enzyme activity applied to the column.
EXAMPLE II
Outdated red blood cells (250 ml) were lucid with 500 ml of distilled water. Insoluble material was removed by tan-genital flow ultrafiltration using a Montana ultrafiltration unit equipped with four 0.45 micron cut-off membranes. Approx-irately 600 ml of filtrate were collected and applied to a 5 cmx 16 cm DEAR - Suffers anion exchange column equilibrated as * trade Mark lZ3Z850 described in Example I. Following sample application, the anion-exchange column was washed with 3 liters of equilibria-lion buffer. Partially purified SOD was eluded from the same DEAR - Suffers column with equilibration buffer containing 0.10M Nail thereby providing an aqueous solution of partially purified SOD.
A column (5 cm x 6 cm) of iminodiacetate-Sepharose 6B
was activated with 50mM aqueous copper chloride solution or 50mM aqueous copper nitrate solution to approximately 3/4 of the bed volume to give an immobilized copper ion column. This column was then used to purify SOD from DEAR - Suffers as described in Example I.
In this case, however, SOD was eluded from the metal chelation affinity column with 20mM sodium citrate buffer at pi 5.0 containing lo Nail. Recovery of enzyme activity, lot-lowing this procedure was about 90% of the total enzyme active fly applied to the column.
EXAMPLE III
Outdated red blood cells (50 ml) were washed twice with saline solution. The centrifugation pellet from the second saline wash was lucid with 100 ml of distilled water. Hero-glob in was removed from the Lucite by chloroform - ethanol treatment and by treatment with solid potassium phosphate (Accord and Fridovich, J. Blot, Chum. 1969, 244, 6049). The resulting ethanolic phase, containing partially purified SOD, was dialyzed overnight against 10mM potassium phosphate buffer at pi 6.4 containing 0.1M Nail. After dialysis the aqueous solution of partially purified SOD was centrifuged and the supernatant subjected to copper chelation affinity cremate-graph as described in Example I.
SUE
EXAMPLE IV
Outdated red blood cells (25 ml) were lucid with unequal volume of distilled water. Most of the hemoglobin was removed from the Lucite by heat treatment at 70C-80C for 15 minutes (Garter et at., Become. J. 1984, 221, 549). Folk lowing centrifugation to remove insoluble material, the sup-ernatant contained partially purified SOD which was dialyzed against 10mM potassium phosphate buffer at pi 6.4 containing lo Nail and further purified by the process of copper Shelley-lion affinity chromatography described in Example I.
EXAMPLE V
A column (2 cm x 2 cam of iminodiacetate Fractogel was activated with 50mM aqueous copper sulfite solution to approx-irately 3/4 of the bed volume to give an immobilized copper ion column. The column was equilibrated at pi 6.4 as described in Example I. Ten ml of SOD solution, from DEAR - Suffers cry-matography, was applied to the immobilized copper ion column followed by 20 ml of equilibration buffer. SOD was eluded from the metal chelation column with 20mM sodium citrate at pi 5 containing lo Nail. Enzyme activity was recovered, completely, following this copper chelation affinity chromatography.
This invention relates to a process for the purifica-lion and production of the enzyme commonly referred to as super oxide decimates (SOD). More particularly, the invent lion relates to a process for the purification and production of super oxide decimates by use of a metal chelating cremate-graphic procedure. The invention is particularly suitable for use in the purification of super oxide decimates from hut man outdated red blood cells.
The invention as claimed herein is a process for puff-lying super oxide decimates which comprises subjecting an aqueous solution containing super oxide decimates which is free from hemoglobin to a metal chelating affinity chromatographic procedure and recovering an aqueous solution containing super-oxide decimates having increased specific activity of super-oxide decimates.
The aqueous solution containing super oxide decimates, which is free from hemoglobin, to be used as starting material, may be conveniently obtained, if desired, from outdated human red blood cells. Such cells, preserved in a suitable medium, such as a citrate-phosphate-dextrose-adenine medium, may be lucid by means of deionized water. The Lucite so obtained may be freed from hemoglobin in a variety of ways, such as a) past sage through an anion exchange chromatographic column using, for example, a cross-linked agrees derivative such as deathly-*
amino ethyl (DEAR) - Suffers, or b) a precipitation technique or c) an affinity technique.
It is preferred to use an anion exchange chromatographic column for the preliminary purification procedure to remove hemoglobin. Any suitable anion exchanger may be used, such as a cross-linked agrees derivative, for example DEAR - Suffers, '~''``"~
* Trade Mark 328S~
in conjunction with an appropriate equilibration buffer which may be for example potassium phosphate at pi 6.4. The condo-lions for separating the super oxide decimates from the hero-glob in on the anion exchange column by elusion may be varied considerably. A buffer with a molar concentration of 0.1M or higher and with a pi of about 5 or higher is a suitable buff for for elusion of the super oxide decimates. Such buffers may be, for example, 0.1M potassium phosphate solution used alone or 10mM potassium phosphate containing either elm sod-I'm chloride or elm potassium chloride. Operation of the column at a temperature of about 4C and at a flow rate of about 150 ml/hour provides an equate in the form of an aqueous solution containing super oxide decimates which is suitable for use in the subsequent metal chelating affinity chromatographic procedure.
As an alternative method for providing the starting mat-trial containing super oxide decimates, it is possible to sub-jet red blood cells to a saline wash followed by a tangential flow ultrafiltration system and rupture of the cells hypotoni-gaily before the Lucite is applied to the anion exchange got-urn. Thus, the red blood cells may be washed with about 10 volumes of saline using a tangential flow ultrafiltration soys-them. The saline wash is carried out twice followed by concern-traction of red blood cells using the same ultrafiltration soys-them. Cells are then ruptured, hypotonically, by the addition of 3 volumes of distilled water. The cell membranes are no-moved from the soluble proteins using the ultrafiltration apt pyrites and filtered Lucite is then applied to the anion ox-change column. The ultrafiltration process removes the bulk of the plasma proteins present in red blood cell concentrate and thus increases the efficiency of the process to provide 1232~350 the starting material in the form of an aqueous solution con-twining super oxide decimates. Another variation of the ultra-filtration process that can be used is the removal of insoluble material from red blood cell lusts by filtration without prior washing of the cells with saline.
The metal chelating affinity chromatographic procedure may be carried out on a suitable column wherein the chelated metal is attached to an insoluble matrix. A suitable matrix may be a cross-linked agrees derivative, a derivative from vinyl polymers or a derivative from cellulose or silica or polyacrylamides. Suitable cross-linked agrees derivatives may be, for example, iminodiacetate-Sepharose 6B or ethylene-diamine-Sepharose or tris-(carboxymethyl)ethylenediamine-Seph-arose. Of these, it is preferred to use iminodiacetate-Sepha-rose 6B as the column of choice. A suitable matrix as a don-ivative from vinyl polymers may be iminodiacetate-Fractogel.
Other matrix columns may be prepared from an iminodiacetate-linked derivative of cellulose, polyacrylamide, silica or sync Thetis polymeric supports.
It is to be understood that the commercially available *
product known as iminodiacetate-Sepharose 6B is a beaded age-rose gel consisting of the chelating agent, iminodiacetic acid, coupled to agrees after epoxy activation, the latter introduce in some cross-linking into the matrix. The product thus has bis-(carboxymethyl)-amino groups coupled to agrees via long, stable, hydrophilic spacer arms as shown by the formula:
agarose-O-CH2CHOHCH2O(CH2)4OCH2CHOHCH2N(CH2COOH)2 The metal to be immobilized on the column is preferably copper and the immobilization procedure may be carried out by applying an aqueous solution of a copper salt to the column.
* Trade Mark A variety of copper salts may be used, such as copper sulfite, copper chloride and copper nitrate. A preferred copper salt is copper sulfite and a 50mM aqueous solution of copper sulfite may be applied to a column of iminodiacetate-Sepharose 6B which has previously been equilibrated with a buffer of loom poles-slum phosphate (pi 6.4) containing Old sodium chloride. A
buffer having a pi from about 6 to about 8 provides suitable conditions for binding the super oxide decimates to the copper chelating column. When the metal chelating affinity column has been equilibrated, the aqueous solution containing super-oxide decimates, prepared as starting material, may be applied to the immobilized copper column. Subsequent elusion provides an aqueous solution containing highly purified super oxide disk mutate showing a specific activity of about 3500 unit/mg. As a suitable eluant, there may be used 20mM sodium citrate buff for at pi 5.0 or 20mM sodium citrate buffer at pi 5.0 contain-in lo sodium chloride.
It will be appreciated by those skilled in the art that other suitable columns and other copper salts, with appropriate buffers, may be used to provide the immobilized copper cheat-in affinity column on which the aqueous solution of super oxide decimates might be applied and thereafter eluded to provide highly purified super oxide decimates.
The process of this invention is particularly valuable when the copper chelating column is prepared from an aqueous solution of copper sulfite applied to a column of iminodiace-tate-Sepharose 6B. The process is further enhanced when the starting material to be used on such a copper chelating aft finite column is an aqueous solution containing super oxide disk mutate which has been obtained as an equate from a cremate-graphic column containing diethylaminoethyl (DEAR) - Suffers ~Z3Z850 to remove hemoglobin from human red blood cell Lucite.
Thus, as a further feature of the invention, as claimed herein there is provided a process for purifying super oxide decimates which comprises subjecting human red blood cell lye sate to an anion exchange chromatographic procedure, wherein the anion exchanger is DEAR - Suffers, to provide an aqueous solution containing super oxide decimates free from hemoglobin and then subjecting the aqueous solution to an immobilized copper ion chromatographic procedure, wherein the immobilized copper ion is prepared from an aqueous solution of a copper salt, such as copper sulfite, copper chloride or copper nix irate on a column of iminodiacetate-Sepharose 6B, and then eluding the column to provide an aqueous solution containing super oxide decimates having increased specific activity of super oxide decimates.
The invention is illustrated by, but not limited by, the following Examples.
EXAMPLE I
The following example describes a procedure for purify-in super oxide decimates (SOD) by chromatography on a column of immobilized copper ions.
Outdated red blood cells in suspension (15 ml), pro-served in citrate-phosphate-dextrose-adenine, were lucid with an equal volume of deionized water. The Lucite so obtained was then applied to a 5 cm x 4 cm column of DEAR - Suffers equilibrated with 10mM potassium phosphate buffer (pi 6.4) at 4C and at a flow rate of 150 ml/hour. The same temperature and flow rate were maintained throughout the remainder of the chromatographic procedure. After the Lucite had been applied, the column was washed with approximately 250 ml of equilibria-lion buffer. Partially purified SOD was then eluded from the ~232~350 DEAR - Suffers column with the same equilibration buffer containing elm sodium chloride thereby providing an aqueous solution of partially purified SOD.
A column (5 cm x 2 cm) of iminodiacetate-Sepharose 6B
was activated with 50mM aqueous copper sulfite solution to approximately 3/4 of the bed volume to give an immobilized copper ion column. Prior to application of the partially pun-flied SOD solution, tide immobilized copper ion column was equip liberated with 10mM potassium phosphate buffer pi 6.4) contain-in 0.1M Nail. The aqueous solution of partially purified SOD,eluted from the DEAR - Suffers column, was applied to the imp mobilized copper ion column and thereafter washed through with 10mM potassium phosphate buffer containing lo Nail. After the effluent reached an absorbency of zero, at 280 no, the column was washed with 50 ml of 10mM potassium phosphate buffer (pi 6.4). Purified SOD was eluded from the copper ion chelates column with 20mM sodium citrate buffer (pi 5.0). Enzyme anti-viny (Holland et at., Anal. Become. 1983, 135, 282) and pro-loin determinations (Lowry et at., J. Blot. Chum. 1951, 193, 265) indicated that the specific activity of the SOD increased about 270-fold as a result of this chromatographic procedure on copper ion iminodiacetate-Sepharose 6B. Recovery of enzyme activity, following this procedure, ranged from 75% to 80% of the total enzyme activity applied to the column.
EXAMPLE II
Outdated red blood cells (250 ml) were lucid with 500 ml of distilled water. Insoluble material was removed by tan-genital flow ultrafiltration using a Montana ultrafiltration unit equipped with four 0.45 micron cut-off membranes. Approx-irately 600 ml of filtrate were collected and applied to a 5 cmx 16 cm DEAR - Suffers anion exchange column equilibrated as * trade Mark lZ3Z850 described in Example I. Following sample application, the anion-exchange column was washed with 3 liters of equilibria-lion buffer. Partially purified SOD was eluded from the same DEAR - Suffers column with equilibration buffer containing 0.10M Nail thereby providing an aqueous solution of partially purified SOD.
A column (5 cm x 6 cm) of iminodiacetate-Sepharose 6B
was activated with 50mM aqueous copper chloride solution or 50mM aqueous copper nitrate solution to approximately 3/4 of the bed volume to give an immobilized copper ion column. This column was then used to purify SOD from DEAR - Suffers as described in Example I.
In this case, however, SOD was eluded from the metal chelation affinity column with 20mM sodium citrate buffer at pi 5.0 containing lo Nail. Recovery of enzyme activity, lot-lowing this procedure was about 90% of the total enzyme active fly applied to the column.
EXAMPLE III
Outdated red blood cells (50 ml) were washed twice with saline solution. The centrifugation pellet from the second saline wash was lucid with 100 ml of distilled water. Hero-glob in was removed from the Lucite by chloroform - ethanol treatment and by treatment with solid potassium phosphate (Accord and Fridovich, J. Blot, Chum. 1969, 244, 6049). The resulting ethanolic phase, containing partially purified SOD, was dialyzed overnight against 10mM potassium phosphate buffer at pi 6.4 containing 0.1M Nail. After dialysis the aqueous solution of partially purified SOD was centrifuged and the supernatant subjected to copper chelation affinity cremate-graph as described in Example I.
SUE
EXAMPLE IV
Outdated red blood cells (25 ml) were lucid with unequal volume of distilled water. Most of the hemoglobin was removed from the Lucite by heat treatment at 70C-80C for 15 minutes (Garter et at., Become. J. 1984, 221, 549). Folk lowing centrifugation to remove insoluble material, the sup-ernatant contained partially purified SOD which was dialyzed against 10mM potassium phosphate buffer at pi 6.4 containing lo Nail and further purified by the process of copper Shelley-lion affinity chromatography described in Example I.
EXAMPLE V
A column (2 cm x 2 cam of iminodiacetate Fractogel was activated with 50mM aqueous copper sulfite solution to approx-irately 3/4 of the bed volume to give an immobilized copper ion column. The column was equilibrated at pi 6.4 as described in Example I. Ten ml of SOD solution, from DEAR - Suffers cry-matography, was applied to the immobilized copper ion column followed by 20 ml of equilibration buffer. SOD was eluded from the metal chelation column with 20mM sodium citrate at pi 5 containing lo Nail. Enzyme activity was recovered, completely, following this copper chelation affinity chromatography.
Claims (33)
1. A process for purifying superoxide dismutase which comprises subjecting an aqueous solution containing said sup-eroxide dismutase which is free from hemoglobin to a metal che-lating affinity chromatographic procedure and recovering an aqueous solution containing superoxide dismutase having in-creased specific activity of superoxide dismutase.
2. The process of claim 1 wherein the metal chelating affinity chromatographic procedure is a copper chelating af-finity chromatographic procedure.
3. The process of claim 1 wherein the metal chelating affinity chromatographic procedure is carried out by use of an immobilized copper ion chromatographic column.
4. The process of claim 1, 2 or 3 wherein the chroma-tographic procedure uses a matrix which is selected from a cross-linked agarose derivative, a derivative from vinyl poly-mers and a derivative from cellulose or silica or polyacryl-amides.
5. The process of claim 1, 2 or 3 wherein the chroma-tographic procedure uses a matrix which is selected from im-inodiacetate-?epharose 6B, ethylenediamine-?epharose, tris-(carboxymethyl)ethylenediamine-?epharose, iminodiacetate-?ractogel and iminodiacetate-derivatives of cellulose or silica or polyacrylamide or synthetic polymeric supports.
6. The process of claim 1, 2 or 3 wherein the chroma-tographic procedure uses a matrix which is iminodiacetate-/epharose 6B.
7. The process of claim 3 wherein the immobilized cop-per ion chromatographic column is prepared from a column of iminodiacetate-?epharose 6B activated with an aqueous solution of a copper salt.
8. The process of claim 7 wherein the copper salt is copper sulphate, copper chloride or copper nitrate.
9. The process of claim 8 wherein the copper salt is used in the form of a 50mM aqueous copper sulphate, copper chloride or copper nitrate solution.
10. The process of claim 7, 8 or 9 wherein the column of iminodiacetate-?epharose 6B is activated with the aqueous copper salt solution to approximately 3/4 of the bed volume.
11. The process of claim 3 wherein the immobilized cop-per ion chromatographic column is equilibrated with a buffer before application of the aqueous solution containing the sup-eroxide dismutase.
12. The process of claim 11 wherein the buffer has a pH of from about 6 to about 8.
13. The process of claim 12 wherein the buffer is po-tassium phosphate or sodium phosphate.
14. The process of claim 13 wherein the buffer is an aqueous 10mM potassium phosphate buffer having a pH of 6.4 and containing 0.1M sodium chloride.
15. The process of claim 1, 2 or 3 wherein the aqueous solution containing superoxide dismutase used as starting mat-erial is obtained from a chromatographic procedure using an anion exchanger.
16. The process of claim 1, 2 or 3 wherein the aqueous solution containing superoxide dismutase used as starting mat-erial is obtained from an anion exchange column.
17. The process of claim 1, 2 or 3 wherein the aqueous solution containing superoxide dismutase used as starting mat-erial is obtained by use of an anion exchange column contain-ing DEAE - ?epharose as the ion exchanger.
18. The process of claim 7, 8 or 9 wherein the aqueous solution containing superoxide dismutase used as starting mat-erial is obtained from a chromatographic procedure using an anion exchanger.
19. The process of claim 7, 8 or 9 wherein the aqueous solution containing superoxide dismutase used as starting mat-erial is obtained from an anion exchange column.
20. The process of claim 7, 8 or 9 wherein the aqueous solution containing superoxide dismutase used as starting mat-erial is obtained by use of an anion exchange column contain-ing DEAE - ?epharose as the ion exchanger.
21. A process for purifying superoxide dismutase which comprises subjecting human red blood cell lysate to a prelim-inary purification procedure to remove hemoglobin therefrom thus providing an aqueous solution containing partially puri-fied superoxide dismutase, subjecting said aqueous solution to a copper chelating affinity chromatographic procedure and there-after obtaining an aqueous solution containing superoxide dis-mutase having increased specific activity of superoxide dis-mutase.
22. A process for purifying superoxide dismutase which comprises subjecting human red blood cell lysate to a prelim-inary purification procedure to remove hemoglobin therefrom thus providing an aqueous solution containing partially puri-fied superoxide dismutase, passing said aqueous solution through an immobilized copper ion chromatographic column and then elut-ing said column to provide an aqueous solution containing sup-eroxide dismutase having increased specific activity of super-oxide dismutase.
23. The process of claim 22 wherein the preliminary purification procedure is carried out by use of an anion ex-change chromatographic procedure.
24. The process of claim 21, 22 or 23 wherein the chro-matographic procedure uses DEAE - ?epharose as the anion ex-changer.
25. The process of claim 21, 22 or 23 wherein the cop-per chromatographic procedure consists of immobilized copper ion on a column of iminodiacetate-?epharose 6B.
26. The process of claim 21, 22 or 23 wherein the cop-per chromatographic procedure uses an immobilized copper ion chromatographic column prepared by the action of an aqueous solution of copper sulphate on said column.
27. The process of claim 21, 22 or 23 wherein the cop-per chromatographic procedure uses an immobilized copper ion chromatographic column prepared by the action of an aqueous copper sulphate solution on a column of iminodiacetate-?epha-rose 6B.
28. A process for purifying superoxide dismutase which comprises subjecting human red blood cell lysate to an anion exchange chromatographic procedure to provide an aqueous sol-ution containing superoxide dismutase free from hemoglobin and then subjecting said aqueous solution to an immobilized copper ion chromatographic procedure and then eluting said column to provide an aqueous solution containing superoxide dismutase having increased specific activity of superoxide dismutase.
29. The process of claim 28 wherein the anion exchange chromatographic procedure uses an anion exchanger which is DEAE - ?epharose.
30. The process of claim 28 wherein the immobilized cop-per ion chromatographic procedure uses a chromatographic col-umn of iminodiacetate-?epharose 6B.
31. The process of claim 28, 29 or 30 wherein the cop-per ion is provided by copper sùlphate, copper chloride or copper nitrate.
32. The process of claim 28, 29 or 30 wherein the im-mobilized copper ion is provided by copper sulphate.
33. The process of claim 28, 29 or 30 wherein the im-mobilized copper ion column is prepared from an aqueous solu-tion of copper sulphate on the column.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000472274A CA1232850A (en) | 1985-01-17 | 1985-01-17 | Purification of superoxide dismutase |
| EP85305900A EP0188053A3 (en) | 1985-01-17 | 1985-08-19 | Purification of superoxide dismutase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000472274A CA1232850A (en) | 1985-01-17 | 1985-01-17 | Purification of superoxide dismutase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1232850A true CA1232850A (en) | 1988-02-16 |
Family
ID=4129594
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000472274A Expired CA1232850A (en) | 1985-01-17 | 1985-01-17 | Purification of superoxide dismutase |
Country Status (2)
| Country | Link |
|---|---|
| EP (1) | EP0188053A3 (en) |
| CA (1) | CA1232850A (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3636208A1 (en) * | 1986-10-24 | 1988-05-05 | Call Hans Peter | METHOD FOR DELIGNIFYING AND WHICH BLEACHING LIGNICELLULOSE-CONTAINING OR LIGNINAL MATERIAL OR LIGNIN BY ENZYMATIC TREATMENT |
| CN1037525C (en) * | 1992-07-30 | 1998-02-25 | 静天玉 | Preparation method for super-oxidate disproportionated enzyme from blood lump |
| DE4239877C1 (en) * | 1992-11-27 | 1994-03-17 | Boehringer Ingelheim Int | Stabilized superoxide dismutase (SOD) composition |
| CN105685619B (en) * | 2014-11-28 | 2018-09-14 | 山东金诃药物研究开发有限公司 | A kind of preparation method of the barley green rich in superoxide dismutase |
| CN104593338A (en) * | 2014-12-31 | 2015-05-06 | 唯美度科技（北京）有限公司 | Method of extracting SOD (superoxide dismutase) from earthworms |
| CN104560904A (en) * | 2014-12-31 | 2015-04-29 | 唯美度科技（北京）有限公司 | Antioxidant enzyme extracted from blood |
| CN109055325B (en) * | 2018-08-28 | 2022-08-16 | 佛山科学技术学院 | Separation and purification method of duck blood erythrocyte SOD |
| CN113231049B (en) * | 2021-05-11 | 2022-11-01 | 南京工业大学 | Cross-linked agarose affinity medium, and preparation method and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK144800C (en) * | 1980-04-21 | 1982-10-25 | Forenede Bryggerier As | PROCEDURE FOR THE EXTRACTION OF ENZYMES, PRIOR CU, ZN SUPEROXIDE DISMUTASE (SOD), CATALASE AND CARBONIC ACID ANHYDRASE, FROM BLOOD |
| JPS6012024B2 (en) * | 1980-08-11 | 1985-03-29 | ヤマサ醤油株式会社 | Method for producing superoxide dismutase |
| JPS58121791A (en) * | 1982-01-14 | 1983-07-20 | Takeda Chem Ind Ltd | Separating and purifying method of superoxide dismutase |
| JPS6012026B2 (en) * | 1982-04-19 | 1985-03-29 | わかもと製薬株式会社 | Purification method of superoxide dismutase |
-
1985
- 1985-01-17 CA CA000472274A patent/CA1232850A/en not_active Expired
- 1985-08-19 EP EP85305900A patent/EP0188053A3/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| EP0188053A2 (en) | 1986-07-23 |
| EP0188053A3 (en) | 1988-04-27 |
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